Beck, Hans Christian

Kramer, Annemarie

Kumar, Abhishek

OceanRep

Quantitative Proteomic Analysis of Anti-cancerous Scopularide production by Scopulariopsis brevicaulis LF580 Annemarie Kramer1), Hans Christian Beck2), Abhishek Kumar3)  1) Kiel Centre for Marine Natural Products, GEOMAR Helmholtz Centre for Ocean Research Kiel, Am Kiel-Kanal 44, 24106 Kiel, Germany 3) Centre for Clinical Proteomics, Department for Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark 2) Department of Genetics & Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Ohlshausenstraße 40, 24098 Kiel ,Germany Protein extraction  Two cell disruption methods were compared, based on grinding in liquid nitrogen and disruption using beads. Adapted protein extraction method using several washing steps, as well as the use of 8 M urea, lead to an increase of the number of identified unique proteins (4759).   Extracting grade of the liquid nitrogen (N) based extraction method:         Integration of further solubilisation steps combined with a predigest into the extraction protocol lead to an increase in coverage. Hence the analyses comprised both, the easy to solve proteins (sn) as well as the precipitating proteins (p). Introduction Within the EU-project MARINE FUNGI (EU FP7, 265926), the marine fungus Scopulariopsis brevicaulis LF580, isolated from the marine sponge Tethya aurantium, was selected for genome analysis as well as for several optimization process including random UV mutagenesis, with respect to the production of the bioactive cyclodepsipeptides. In order to gain a deeper understanding of the hitherto uncharacterized molecular mechanisms underlying the production of scopularide A and B by Scopulariopsis brevicaulis LF580, we applied quantitative proteomics to compare the proteomes extracted from the wild type strain and the mutant strain M26. Using iTRAQ-based quantitative proteomic approach will provide novel biological information useful for the targeted optimization of scopularide production by Scopulariopsis brevicaulis LF580.  References [1] Yu, Z; Lang, G; Kajahn, I; Schmaljohann, R; Imhoff, JF J. Nat. Prod. 2008, 71, 1052–1054 [2] Beck HC; Petersen J; Felthaus O; Schmalz G; Morsczeck C Neurochem Res 2011, 36: 2002-2007 p: 90.7% sn: 79.5% p+sn: 4411 Scopularide A/B [1]: Scopularide A: R = C2H5 Scopularide B: R = H 44 41 37 33 33 26 19 17 9 9 6 6 4 4 3 2 40 24 8 8 7 4 67 62 61 52 34 25 24 18 15 11 7 7 6 6 6 5 5 4 4 4 3 3 3 3 3 3 3 2 2 oxidoreductase activity ion binding hydrolase activity organic cyclic compound binding heterocyclic compound binding transferase activity small molecule binding cofactor binding protein binding lyase activity substrate-specific transporter … transmembrane transporter activity sequence-specific DNA binding … isomerase activity metal cluster binding peroxidase activity cell part membrane-bounded organelle membrane part organelle part protein complex non-membrane-bounded organelle single-organism metabolic process cellular metabolic process organic substance metabolic … primary metabolic process nitrogen compound metabolic … catabolic process single-organism cellular process biosynthetic process regulation of biological process establishment of localization methylation cellular component organization single-organism developmental … regulation of molecular function cellular response to stimulus response to stress anatomical structure development single-multicellular organism … cellular process involved in … reproductive process sexual reproduction multicellular organismal … cellular localization response to chemical stimulus cellular developmental process regulation of biological quality macromolecule localization asexual reproduction reproduction of a single-celled … 37 35 35 20 17 16 12 7 7 6 6 4 3 2 2 42 27 16 15 15 7 50 45 45 31 28 24 21 16 14 7 6 6 5 4 3 3 3 3 3 3 3 2 ion binding organic cyclic compound … heterocyclic compound … small molecule binding hydrolase activity transferase activity oxidoreductase activity substrate-specific … transmembrane transporter … ligase activity structural constituent of … sequence-specific DNA … cofactor binding lyase activity peroxidase activity cell part membrane-bounded organelle non-membrane-bounded … membrane part organelle part protein complex organic substance metabolic … primary metabolic process cellular metabolic process nitrogen compound … single-organism metabolic … biosynthetic process single-organism cellular … establishment of localization cellular component biogenesis regulation of biological … catabolic process cellular response to stimulus response to stress cellular localization single-organism … multicellular organismal … single-multicellular organism … methylation anatomical structure … cellular component … single organism signaling macromolecule localization Molecular  Function Cellular Compartment Biological Process Down-regulated proteins Up-regulated proteins Up-regulated proteins Down-regulated proteins I: Gene ontology (GO) terms of three categories II: KEGG pathways analyses 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Phenylalanine metabolism Fatty acid degradation Folate biosynthesis Glycine, serine and threonine … Phenylpropanoid biosynthesis Porphyrin and chlorophyll … Purine metabolism Amino sugar and nucleotide … Aminoacyl-tRNA biosynthesis Benzoate degradation Biosynthesis of unsaturated … Drug metabolism - other … Ethylbenzene degradation Fatty acid elongation Geraniol degradation Glycerophospholipid … Isoquinoline alkaloid … Lipoic acid metabolism Naphthalene degradation Other types of O-glycan … Pyrimidine metabolism Starch and sucrose metabolism Thiamine metabolism Tropane, piperidine and … Tyrosine metabolism Valine, leucine and isoleucine … alpha-Linolenic acid metabolism beta-Alanine metabolism 7 7 6 6 6 5 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Butanoate metabolism Propanoate metabolism Alanine, aspartate and glutamate … Citrate cycle (TCA cycle) Glyoxylate and dicarboxylate … Nitrogen metabolism Carbon fixation pathways in … Glycolysis / Gluconeogenesis Methane metabolism Oxidative phosphorylation Pentose phosphate pathway Purine metabolism Pyruvate metabolism Starch and sucrose metabolism Toluene degradation Arginine and proline metabolism Carbon fixation in photosynthetic … Fructose and mannose metabolism Glutathione metabolism Glycine, serine and threonine … Tryptophan metabolism Tyrosine metabolism Amino sugar and nucleotide sugar … Aminobenzoate degradation Caffeine metabolism Cyanoamino acid metabolism Drug metabolism - other enzymes Galactose metabolism Glycerophospholipid metabolism Sphingolipid metabolism Styrene degradation Valine, leucine and isoleucine … beta-Alanine metabolism Arachidonic acid metabolism Benzoate degradation Chlorocyclohexane and … Cysteine and methionine metabolism D-Arginine and D-ornithine … Fluorobenzoate degradation Glycosaminoglycan degradation Glycosphingolipid biosynthesis - … Inositol phosphate metabolism Metabolism of xenobiotics by … N-Glycan biosynthesis One carbon pool by folate Other glycan degradation Penicillin and cephalosporin … Phenylalanine metabolism Pyrimidine metabolism Streptomycin biosynthesis Taurine and hypotaurine metabolism Ubiquinone and other terpenoid-… Valine, leucine and isoleucine … Various types of N-glycan … Qualified proteins:   28 54 114 iTRAQ labelling: 3526 unique proteins were identified using iTRAQ labelling combined with nano-LC-MSMS analysis. 318 (9%) proteins showed a regulation, based on the normalization of the ratio against the median peptide ratio. 5.3% were down-regulated, 3.7% represent an up-regulation.  Gene ontology and KEGG pathway analyses  Summary of gene ontology (GO) terms in three categories (I) and of KEGG pathway analyses (II) for down- and up-regulated proteins, based on iTRAQ labelling. Annotational analyses were performed using BLAST2GO tool. iTRAQ labelling HILIC separation nano-LC MS/MS analysis [2] Data analysis: MASCOT-SEQUEST /  annotated genome of LF580 Identification of significant regulated proteins by statistical analysis Gene ontology and KEGG pathway analyses of  regulated proteins Cultivation in triplicates of WT and M26 Two methods of cell disruption: liquid nitrogen and beads Protein extraction Conclusion  The development of an efficient protein extraction method enabled us to extract increased and comparable amounts of proteins from the Scopulariopsis brevicaulis LF580 strain and its UV mutant strain (Scopulariopsis brevicaulis LF580-M26).  Gene ontology analysis of the differentially regulated proteins in the mutant strain as compared to the wild type strain demonstrated an enrichment of proteins and pathways involved in growth and metabolism.  KEGG pathway analysis demonstrated that the down-regulated proteins were associated with enzymes of the TCA cycle,  butanoate and propanoate metabolism, and the metabolism of the TCA-derived amino acids glutamate and aspartate, whereas the up-regulated proteins were associated with the biosynthesis of the oxaloacetate-derived amino acids, serine, glycine, and threonine, fatty acid degradation, and phenylalanine metabolism. Cultivation-based characterization of mutant strain LF580-M26  The UV-mutant strain M26 of Scopulariopsis brevicaulis LF580 exhibited a modified morphological growth (A) in liquid medium, as well as a faster biomass production resulting in a higher yield of scopularide A and B (B).  3 4 5 6 7 8 0 1 10 100 0 24 48 72 96 120 144 pH-Value Log  biomass [mg/mL] Cultivation time [h] A: Pellet formation and shape of WT and M26: Cultivation in 300 mL Erlenmeyer flasks containing 100 mL WSP30-medium B: Time-series of WT and M26: carried out in 300 mL Erlenmeyer flasks containing 100 mL WSP30-medium: Determination of biomass of WT (■) and M26 (●), as well as pH of WT ( ◆ ) and M26 ( ▲ ) in triplicates. B A 

Quantitative proteomics of the regulation patterns in scopularide A and B production by a marine Scopulariopsis brevicaulis strain LF580

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